Optically active β-aminocarboxylic acids occur in natural substances such as alkaloids and antibiotics. The isolation of these amino acids is of interest because of their importance as essential intermediates in the preparation of pharmaceuticals (see, e.g., Juaristi, et al., Curr. Med. Chem. 6:983–1004 (1999)). Both the free form of optically active β-aminocarboxylic acids and their derivatives have interesting pharmacological effects and can also be used in the synthesis of modified peptides.
Methods for preparing β-aminocarboxylic acids include conventional racemate resolution by means of diastereomeric salts (see e.g., Boesch et al., Org. Proc. Res. Developm. 5:23–27 (2001)) and, in particular, the diastereoselective addition of lithium phenylethylamide (Abdel-Magid, et al., Curr. Med. Chem. 1999, 6:955–970 (1999)). The latter method has been intensively researched and, despite numerous disadvantages, is generally considered the preferred method of preparation. One of its drawbacks is that, unlike catalytic asymmetrical methods, stoichiometric amounts of a chiral reagent are needed. Expensive and hazardous auxiliary agents, e.g., n-butyllithium, are also needed to activate the stoichiometric reagent by deprotonation. In addition, the reaction must be performed at a low temperature, e.g., approximately −70° C., for satisfactory stereoselectivity, and this imposes restrictions on reactor material, leads to additional costs and entails high energy consumption.
Although biocatalytic methods of preparing optically active β-aminocarboxylic acids have not found widespread use, they offer both economic and ecological advantages. Stoichiometric amounts of a chiral reagent are unnecessary and, instead, small, catalytic amounts of enzymes are used that are natural and environmentally-friendly. Moreover, in contrast to a multiplicity of synthetic metal-containing catalysts, biocatalysts do not require the presence of a metal-containing, and, consequently, toxic feedstock.
The enantioselective N-acylation of β-aminocarboxylic acids has previously been reported. For example, Kanerva et al. describe the enantioselective N-acylation of ethyl esters of various cycloaliphatic β-aminocarboxylic acids with 2,2,2-trifluoroethyl ester in organic solvents using lipase SP 526 from Candida antarctica or lipase PS from Pseudomonas cepacia as biocatalyst (Tetrahedron: Asymmetry 7(6):1707–1716 (1996)).
Sánchez et al. studied the biocatalytic racemate resolution of (±)-ethyl 3-aminobutyrate with lipase from Candida antarctica via the preparation of N-acetylated β-aminocarboxylic esters (Tetrahedron: Asymmetry 8(1):37–40 (1997)).
EP-A-8 890 649 discloses a process for preparing optically active amino acid esters from a racemic mixture by enantioselective acylation with a carboxylic ester in the presence of a hydrolase selected from the group comprising amidase, protease, esterase and lipase. The unreacted enantiomers of the amino acid esters are subsequently isolated.
WO-A-98/50575 describes a process for obtaining a chiral β-aminocarboxylic acid or its corresponding ester by bringing a racemic β-aminocarboxylic acid, an acyl donor and penicillin G acylase into contact with one another under conditions for stereoselectively acylating one enantiomer of the racemic β-aminocarboxylic acid and under which the other enantiomer is substantially not reacted. A chiral β-aminocarboxylic acid is thus obtained. The reverse reaction sequence has also been studied (Soloshonok, et al., Synlett:339–341 (1993); Soloshonok, et al., Tetrahedron: Asymmetry 5:1119–1126 (1994); Soloshonok, et al., Tetrahedron: Asymmetry 6:1601–1610 (1995); Cardillo, et al., Eur. J Org. Chem. 155–161 (1999)). A disadvantage of this process is that it requires a difficult work-up of the product mixture after enantioselective hydrolysis. After isolating the free β-aminocarboxylic acid, a mixture of phenylacetic acid and N-phenylacetyl-β-aminocarboxylic acid is obtained that is difficult to separate.
A method for obtaining enantiomer-enriched carboxylic acids involves reaction with lipases and, in U.S. Pat. No. 5,518,903, this method was applied to N-protected β-amino acid esters, with varying success. Although the corresponding benzyl ester of racemic N-butoxycarbonyl-β-aminobutyric acid was resolved highly enantioselectively, the remaining methyl esters or n-butyl esters used gave ee values of not more than 70% ee. In this connection, it should be noted that, apparently, going from a methyl ester to a corresponding n-butyl ester is accompanied by an impairment of the ee value of the acid prepared. Thus, starting from the n-butyl ester of N-Boc-β-aminobutyric acid, ester hydrolysis with the enzyme lipase from Asahi results after 8 days in an ee value of the corresponding acid of 45% ee in a yield of 37%. With the lipase PS supplied by Amano, a compound enriched to 61% ee is obtained in the same reaction with a yield of 41% within 7 days. In comparison, the corresponding methyl ester yields 70% ee.
From recently published results it may be inferred that the ester hydrolysis of aromatic β-amino acid ethyl esters at a pH of 8 with the lipase PS supplied by Amano takes place with acceptable yields and very good enantiomeric excesses (Faulconbridge et al., Tetrahedron Letters 41:2679–81 (2000)). The product is obtained with an enantiomeric purity of up to 99% ee, but the synthesis, which was performed exclusively in aqueous suspension, is associated with some disadvantages. Although the crystallization is selective under these conditions, the reaction per se results, as documented in Comparison Example 2, in lower ee values, 85.1%. This means a loss in yield due to the formation of the undesirable enantiomer and suggests that ee values may easily also drop below 99% ee or even below 98% ee as a function of slight process fluctuations or because of altered crystallization conditions. An ee value greater than 98% ee, and preferably greater than 99% ee, is, however, often a requirement for pharmaceutical applications. In addition, performance in purely liquid medium would be desirable in order, for example, to be able to ensure good isolation results by means of ultrafiltration. Optimally, a high ee value should likewise be produced in this step.
An enzymatic hydrolysis in the presence of single-phase reaction media using organic solvents was reported by Nagata et al. (Tetrahedron: Asymmetry 9:4295–4299 (1998)). In that case, a cyclic β-amino acid ester was used. The best results (enantioselectivities of 94% ee and conversions of 50% with a reaction time of 20 h) were achieved using a solvent mixture composed of acetone (90%) and water (10%). Poorer results were achieved with lower proportions of water. In general, the use of readily water-soluble solvents has proved superior compared with sparingly water-miscible solvents. Thus, diisopropyl ether saturated with water and 20% acetone, produces an ee value of only 58% ee.